Protective hydrocolloid for active ingredients

ABSTRACT

Partially deamidated rice endosperm protein or rice endosperm protein which is partially conjugated with mono-, di-, oligo- or polysaccharides is used as novel protective hydrocolloid for fat-soluble active ingredients and/or fat-soluble colorants. The present invention further includes compositions comprising that rice endosperm protein and at least one fat-soluble active ingredient/colorant, as well as their manufacture, that rice endosperm protein itself and its manufacture. These compositions are used for the enrichment, fortification and/or coloration of food, beverages, animal feed, personal care or pharmaceutical compositions. The present invention is directed to theses uses and to food, beverages, animal feed, personal care and pharmaceutical compositions containing such a rice endosperm protein and such a composition, respectively.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation of application Ser. No. 11/729,981,filed Mar. 30, 2007, the specification of which is incorporated hereinby reference.

PROTECTIVE HYDROCOLLOID FOR ACTIVE INGREDIENTS

The present invention is directed to the use of rice endosperm proteinas novel protective hydrocolloid for fat-soluble active ingredientsand/or fat-soluble colorants, whereby the rice endosperm protein ispartially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidated.Moreover, the present invention is directed to compositions comprisingthat partially conjugated or partially deamidated rice endosperm proteinand at least one fat-soluble active ingredient and/or fat-solublecolorant and to their manufacture, as well as to that partiallyconjugated or partially deamidated rice endosperm protein itself and itsmanufacture. The present invention is further directed to the use ofsuch compositions for the enrichment, fortification and/or coloration offood, beverages, animal feed, personal care or pharmaceuticalcompositions, and to food, beverages, animal feed, personal care andpharmaceutical compositions containing such a partially conjugated orpartially deamidated rice endosperm protein and such a composition,respectively.

Active ingredients, especially fat-soluble active ingredients orfat-soluble colorants, are often not added as such to food, beverages,animal feed, personal care and pharmaceutical compositions, but in formof formulations of the active ingredient in a hydroprotective colloidfor reasons of enhancing properties such as chemical stability,(water-)solubility, free-flowing and controlled release etc. Knownhydroprotective colloids are e.g. gelatine of different origin (poultry,bovine, pork, fish) and starch. Since hydroprotective colloids of animalorigin are often not desired for religious or allergenic reasons andstarch-based hydroprotective colloids might have low preference forconsumers who are interested in gluten and corn-free products there isan on-going need for alternative hydroprotective colloids.

Rice endosperm proteins are recognized as nutritional and hypoallergenicand can, thus, be a suitable alternative source of protectivehydrocolloid for formulations of active ingredients. However, highinsolubility and poor functionality of rice endosperm protein at neutralpH limits its industrial application as a functional ingredient in foodand pharmaceuticals products. The present invention overcomes theselimitations and incorporates the rice endosperm protein which ispartially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidated as aprotective hydrocolloid for formulations of fat-soluble activeingredients and/or fat-soluble colorants.

Rice proteins rank high in nutritional quality in comparison to othercereals including corn and wheat, and are therefore perceived to haveimmense potential uses as food ingredients. Cereal grain proteins arerich in the essential amino acids cysteine and methionine. Lysine is theprimary limiting amino acid in cereal proteins, but rice contains morelysine (3.8 g/16 g N) than other cereal proteins (wheat 2.3, corn 2.5g/16 g N) (see reference 4 cited below). Although rice is generallyregarded as having the lowest protein content (7.3%) among the commongrains (wheat 10.6%, corn 9.8%, barley 11.0%, millet 11.5%), the netprotein utilization of rice protein (73.8%) is the highest among thecereal grains (wheat 53.0%, corn 58.0%, barley 62.0%, millet 56.0%).

Compared with other cereal proteins, isolation of rice protein isdifficult and therefore costly. The predominant rice protein, glutelin,is hydrophobic and is cross-linked with disulfide bonds. The extractedproteins are highly insoluble in nature and the conditions used inprotein isolation further decrease their solubility, and thus havelimited application as a functional ingredient. High-protein riceproducts can be obtained from rice flour by alkali extraction followedby precipitation at the isoelectric pH of the protein.Starch-hydrolyzing enzymes such as alpha-amylase, glucoamylase, andpullulanase are often used to separate proteins in rice flour bysolubilizing and removing starch. In addition to starch hydrolyzingenzymes, cellulase and hemicellulase enzymes have been used to furtherincrease the protein content in rice protein concentrate. However,information on suitable extraction methods and functionalities of suchisolates is limited. Efficient extraction methods using approved foodgrade enzymes and chemicals are essential for commercial production andapplication of rice protein.

This need is fulfilled by the compositions of the present inventionwhich comprise a rice endosperm protein which is partially conjugatedwith mono-, di-, oligo- or polysaccharides (especially with mono- orpolysaccharides) or partially deamidated and a fat-soluble activeingredient and/or fat-soluble colorant.

BACKGROUND INFORMATION

-   1. Approved Methods of the American Association of Cereal Chemists,    8th Ed. AACC methods 1990, 44-16 and 46-12. The Association: St.    Paul, Minn.-   2. Achouri, A., Joyce Irene Boye, J. I., Yaylayan V. A., And    Yeboah, F. K.: Functional Properties of Glycated Soy 11S Glycinin. J    Food Sci. 2005, 70 (4), p. 269.-   3. Baniel. A., Caer. D., Colas, B. and Gueguen, J.: Functional    Properties of Glycosylated Derivatives of the 11s Storage Protein    from Pea (Pisum sativum L.). J. Agric. Food Chem. 1992, 40, p.    200-205.-   4. Bera M B, Mukherjee R K.: Solubility, emulsifying, and foaming    properties of rice bran protein concentrates. J. Food Sci. 1989,    54(1), p. 142-145.-   5. Cabra, V. Arreguin, R. Vazquez-Duhalt, R. Farres, A.: Effect of    Alkaline Deamidation on the Structure, Surface Hydrophobicity, and    Emulsifying Properties of the Z19 alpha-Zein J. Agric. Food Chem    2007, 55, p. 439-445.-   6. Kato, A., Sasaki, Y., Furuta, R., and Kobayashi, K.: Functional    protein polysaccharide conjugate prepared by controlled dry-heating    of ovalbumin-dextran mixtures. Agric. Biol. Chem. 1990, 54, p.    107-112.-   7. Kato, A., Shimokawa, K., and Kobayashi, K.: Improvement of the    functional properties of insoluble gluten by Pronase digestion    followed by dextran conjugation. J. Agric. Food Chem. 1991, 39, p.    1053-1056.-   8. Kato, Y., Aoki, T., Kato, N., Nakamura, R., and Matsuda, T.:    Modification of ovalbumin with glucose-6-phosphate by amino-carbonyl    reaction. Improvement of protein heat stability and emulsifying    activity. J. Agric. Food Chem. 1995, 43, p. 301-305.-   9. Kinsella, J. E.: Functional properties of proteins in foods: a    survey. Crit. Rev Food Sci. Nutr. 1976, 8(4), p. 19-80.-   10. Nakamura S, Kato A, Kobayashi K.: Bifunctional    lysozyme-galactomannan conjugate having excellent emulsifying    properties and antibacterial effects. J Agric Food Chem 1992, 40, p.    735-9.-   11. Nakamura, S., Saito, M., Goto, T., Saeki, H., Ogawa, M., Gotoh,    G., Gohya, Y., and Hwang, J.-K.: Rapid formation of biologically    active neoglycoprotein from lysozyme and xyloglucan hydrosylates    through naturally occurring Maillard reaction. J. Food Sci. Nutr.    2000, 5, p. 65-69.-   12. Nielsen, P. M., Petersen, D., and Dambmann, C.: Improved method    for determining food protein degree of hydrolysis. J. Food Sci.    2001, 66 (5), 642-646.-   13. Nielson, P. M.: Functionality of protein hydrolysates. In:    Damodaran S, Paraf A, editors. Food proteins and their applications.    1st ed. New York: Marcel Dekker Inc. 1997, p. 443-72.-   14. Oliver, C. M., Melton, L. D., & Stanley, R. A.: Creating    proteins with novel functionality via the Maillard reaction: A    review. Critical Reviews in Food Science and Nutrition 2006, 46, p.    337-350.-   15. Paraman, I., Hettiarachchy, N. S., Schaefer, C., and Beck. M.    I.: Hydrophobicity, Solubility, and Emulsifying Properties of Enzyme    modified Rice Endosperm Protein. Submitted to Cereal chem. 2007,    Manuscript ID. CC 10-06-0125-   16. Pearce K N, Kinsella J E.: Emulsifying properties of proteins:    evaluation of a turbidimetric technique. J Agric Food Chem 1978,    26, p. 716-722.-   17. SAS (Statistical Analysis System). 2002. JMP® User's Guide,    Version 5. SAS Institute Inc. Cary, N.C.

18. Schwenke K. D.: Enzyme and Chemical Modification of Proteins.Chapter 13 in “Food Proteins and their Applications” edited by A.Damodaran and A Paraf, 1997, Marcel Dekker, Inc., New York, USA.

-   19. Wen, T. N., and Luthe, D. S.: Biochemical characterization of    rice glutelin. Plant Physiol. 1985, 78, p. 172-177.

DETAILED DESCRIPTION OF THE INVENTION Compositions of the PresentInvention

The compositions of the present invention may be solid compositions,i.e. stable, water-soluble or water-dispersible powders, or they may beliquid compositions, i.e. aqueous colloidal solutions or oil-in-waterdispersions of the aforementioned powders. The stabilised oil-in-waterdispersions, which may be oil-in-water emulsions or may feature amixture of suspended, i.e. solid, particles and emulsified, i.e. liquid,droplets, may be prepared by the methods described below or by ananalogous manner.

More specifically, the present invention is concerned with stablecompositions in powder form comprising one or more fat-soluble activeingredient(s) and/or one or more fat-soluble colorant(s) in a matrix ofa rice endosperm protein which is partially conjugated with mono-, di-,oligo- or polysaccharides (especially with mono- or polysaccharides) orpartially deamidated.

Preferably the amount of that rice endosperm protein which is partiallyconjugated with mono-, di-, oligo- or polysaccharides (especially withmono- or polysaccharides) or partially deamidated is from 1 to 70weight-%, more preferably from 5 to 50 weight-%, even more preferablyfrom 10 to 40 weight-%, most preferably from 10 to 20 weight-% (with 20weight-% being the most preferred one) and/or the amount of thefat-soluble active ingredient and/or fat-soluble colorant is from 0.1 to90 weight-%, preferably from 1 to 80 weight-%, more preferably from 1 to20 weight-%, based on the total amount of the composition. If additionaladjuvants and/or excipients such as tocopherol and/or ascorbyl palmitateare present, they are present in an amount of from 0.01 to 50 weight-%,preferably in an amount of from 0.1 to 30 weight-%, more preferably inan amount of from 0.5 to 10 weight-%, based on the total amount of thecomposition.

Rice Endosperm Protein which is Partially Conjugated with Mono-, Di-,Oligo- or Polysaccharides (Especially with Mono- or Polysaccharides) orPartially Deamidated

In preferred embodiments of the present invention the rice endospermprotein is a modified rice endosperm protein whose manufacture isdescribed below.

An especially preferred rice protein is one obtained by the followingsteps: alkaline extraction, (enzymatically modification, especially withAlkalase), partial cross-linking with at least one compound selectedfrom the group consisting of mono-, di-, oligo- and polysaccharides(especially from the group consisting of mono- and polysaccharides),centrifugation and ultra-filtration. A further especially preferred riceprotein is one obtained by the following steps: alkaline extraction,(enzymatically modification, especially with Alkalase), partialdeamidation, centrifugation and ultra-filtration.

If needed for the further use the thus obtained modified rice endospermprotein may also be dried.

In preferred embodiments of the invention the used rice endospermproteins which are partially conjugated with mono-, di-, oligo- orpolysaccharides (especially with mono- or polysaccharides) or partiallydeamidated have an emulsion activity of ≧0.2, preferably of ≧0.45, morepreferably of ≧0.5, even more preferably of from 0.5 to 1.0. Thedetermination of the emulsion activity is described in example 1. Thepresent invention refers also to these rice endosperm proteins which arepartially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidatedthemselves.

The amount of the rice endosperm protein which is partially conjugatedwith mono-, di-, oligo- or polysaccharides (especially with mono- orpolysaccharides) or partially deamidated may be in the range of from 1to 70 weight-%, preferably in the range of from 5 to 50 weight-%, morepreferably in the range of from 10 to 40 weight-%, most preferably inthe range of from 10 to 20 weight-%, based on the total weight of thecomposition as disclosed below.

Fat-Soluble Active Ingredient and/or Fat-Soluble Colorant

The fat-soluble active ingredients are preferably those ingredients witha pharmacological effect or those providing health benefits to the humanor animal body in general. “Fat-soluble” (fat-soluble activeingredient/fat-soluble colorant) in the context of the present inventionmeans that the compound is hardly soluble in water at room temperatureand at atmospheric pressure.

The fat-soluble active ingredient and/or the fat-soluble colorant ispreferably selected from the group consisting of carotenes andstructurally related polyene compounds, fat-soluble vitamins, coenzymeQ10, polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) and esters thereof (such as the ethyl estersor the triglycerides (containing the same or different fatty acids)),mono-, di-, triglycerides rich in polyunsaturated fatty acids,fat-soluble UV-A filters, UV-B filters, as well as their physiologicallyacceptable derivatives such as their esters, especially with C₁₋₂₀carbonic acids, and any mixtures of them.

The most preferred fat-soluble vitamins are Vitamin A or E.

Preferred examples of the carotenes and structurally related polyenecompounds are carotenoids such as α-carotene, β-carotene,8′-apo-β-carotenal, 8′-apo-β-carotenoic acid esters such as the ethylester, canthaxanthin, astaxanthin, lycopene, lutein, zeaxanthin,crocetin, α-zeacarotene, β-zeacarotene, as well as their physiologicallyacceptable derivatives such as their esters, especially with C₁₋₂₀carbonic acids, and any mixtures of them.

The most preferred carotenoid is β-carotene.

The term “β-carotene” encompasses the all-cis as well as the all-transisomers and all possible mixed cis-trans-isomers. The same applies forthe other carotenoids.

The term “zeaxanthin” encompasses the natural R,R-zeaxanthin, as well asS,S-zeaxanthin, meso-zeaxanthin and any mixture of them. The sameapplies for lutein.

The fat-soluble active ingredients may be of natural origin, i.e.isolated/extracted from plants, purified and/or concentrated, as well asthose synthesized by chemical and/or microbiological (fermentative)routes.

The amount of the fat-soluble active ingredient and/or the fat-solublecolorant may be in the range of from 0.1 to 90 weight-%, preferably inthe range of from 1 to 80 weight-%, more preferably in the range of from1 to 20 weight-%, based on the total weight of the composition asdisclosed below.

Further Components

Beside the active ingredient and the rice endosperm protein which ispartially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidated thecompositions of the present invention may preferably additionallycontain at least one water-soluble antioxidant and/or fat-solubleantioxidant. The amount of the water-soluble antioxidant and/orfat-soluble antioxidant may be in the range of from 0.1 to 10.0weight-%, preferably in the range of from 0.5 to 5.0 weight-%, morepreferably in the range of from 0.5 to 3.0 weight-%, based on the totalweight of the composition.

The water-soluble antioxidant may be for example ascorbic acid or a saltthereof, preferably sodium ascorbate, watersoluble polyphenols such ashydroxytyrosol and oleuropein aglycon; epigallocatechingallate (EGCG) orextracts of rosemary or olives.

The fat-soluble antioxidant may be for example a tocopherol, e.g.dl-α-tocopherol (i.e. synthetic tocopherol), d-α-tocopherol (i.e.natural tocopherol), β- or γ-tocopherol, or a mixture of two or more ofthese; butylated hydroxytoluene (BHT); butylated hydroxyanisole (BHA);ethoxyquin, propyl gallate; tert. butyl hydroxyquinoline; or6-ethoxy-1,2-di-hydroxy-2,2,4-trimethylquinoline (EMQ), or an ascorbicacid ester of a fatty acid, preferably ascorbyl palmitate or stearate.

The compositions of the present invention may further contain aco-emulgator selected from the group consisting of mono- anddiglycerides of fatty acids, polyglycerol esters of fatty acids,lecithins; N-acylated amino acids and derivatives thereof, N-acylatedpeptides with an alkyl or alkenyl radical, and salts thereof; alkyl oralkenyl ether or ester sulfates, and derivatives and salts thereof;polyoxyethylenated alkyl or alkenyl fatty ethers or esters;polyoxyethylenated alkyl or alkenyl carboxylic acids and salts thereof;N-alkyl or N-alkenyl betaines; alkyltrimethylammonium oralkenyltrimethylammonium and salts thereof; polyol alkyl or alkenylether or ester; and mixtures thereof.

Preferred examples of polyol alkyl or alkenyl ethers or esters aresorbitan alkyl or alkenyl esters polyoxyethylenated with at least 20units of ethylene oxide, such as sorbitan palmitate 20 EO or Polysorbate40 marketed under the tradename Montanox 40 DF by the company Seppic,sorbitan laurate 20 EO or Polysorbate 20 marketed under the tradenameTween 20 by the company ICI, and sorbitan monostearate.

The amount of the co-emulgator may be in the range of from 0 to 90weight-%, preferably in the range of from 0 to 50 weight-%, morepreferably in the range of from 0 to 20 weight-%, based on the totalweight of the composition.

The formulations according to the present invention may further bepressed into tablets, whereby one or more excipients and/or adjuvantsselected from the group consisting of monosaccharides, disaccharides,oligosaccharides and polysaccharides, glycerol, and triglycerides, maybe added.

Preferred examples of mono- and disaccharides which may be present inthe compositions of the present invention are sucrose, invert sugar,xylose, glucose, fructose, lactose, maltose, saccharose and sugaralcohols.

Preferred examples of the oligo- and polysaccharides are starch,modified starch and starch hydrolysates. Preferred examples of starchhydrolysates are dextrins and maltodextrins, especially those having therange of 5 to 65 dextrose equivalents (DE), and glucose syrup,especially such having the range of 20 to 95 DE. The term “dextroseequivalent” (DE) denotes the degree of hydrolysis and is a measure ofthe amount of reducing sugar calculated as D-glucose based on dryweight; the scale is based on native starch having a DE close to 0 andglucose having a DE of 100.

The triglyceride is suitably a vegetable oil or fat, preferably cornoil, sunflower oil, soybean oil, safflower oil, rapeseed oil, peanutoil, palm oil, palm kernel oil, cotton seed oil, olive oil or coconutoil.

The amount of the excipient(s) and/or adjuvant(s) may be in the range offrom 0.1 to 50 weight-%, preferably in the range of from 0.1 to 30weight-%, more preferably in the range of from 0.5 to 10 weight-%, basedon the total weight of the composition.

Solid compositions may in addition contain an anti-caking agent, such assilicic acid or tricalcium phosphate and the like, and up to 10weight-%, as a rule 2 to 5 weight-%, of water.

The amount of the anti-caking agent may be in the range of from 0 to 5weight-%, preferably in the range of from 0 to 3 weight-%, morepreferably in the range of from 0.2 to 3.0 weight-%, based on the totalweight of the composition.

Manufacture of the Composition

An object of the present invention is also a process for the manufactureof the composition of the present invention which comprises thefollowing steps:

I) preparing an aqueous solution or colloidal solution of a riceendosperm protein which is partially conjugated with mono-, di-, oligo-or polysaccharides (especially with mono- or polysaccharides) orpartially deamidated, (manufacture of such a rice endosperm protein isdescribed below),II) optionally adding at least a water-soluble excipient and/or adjuvantto the solution prepared in step I),III) preparing a solution or dispersion of at least a fat-soluble activeingredient and/or fat-soluble colorant, and optionally at least afat-soluble adjuvant and/or excipient,IV) mixing the solutions prepared in step I) to III) with each other,V) homogenising the thus resulting mixture,VI) optionally adding a cross-linking agent for partially cross-linkingsaid rice endosperm protein which is partially conjugated with mono-,di-, oligo- or polysaccharides (especially with mono- orpolysaccharides) or partially deamidated,VIa) optionally submitting the mixture resulting after having performedstep VI) to enzymatic treatment or heat treatment to partiallycross-link the rice endosperm protein which is partially conjugated withmono-, di-, oligo- or polysaccharides (especially with mono- orpolysaccharides) or partially deamidated;VII) optionally converting the dispersion obtained in step V) and/or VI)into a powder,VIII) optionally drying the powder obtained in step VII),IX) optionally submitting the dry powder to heat treatment or toenzymatic treatment to cross-link the (modified) rice endosperm protein,with the proviso that only step VIa) or step IX) is carried out, but notboth, when step VI) is carried out.

Step I

This step is simply performed by adding water to the rice endospermprotein which is partially conjugated with mono-, di-, oligo- orpolysaccharides (especially with mono- or polysaccharides) or partiallydeamidated (manufacture see below) or vice versa, optionally understirring. Alternatively homogenization may be possible viaultrasonication.

Preferably the rice endosperm protein which is partially conjugated withmono-, di-, oligo- or polysaccharides (especially with mono- orpolysaccharides) or partially deamidated is used with the preferences asdescribed above and below.

Step II

Water-soluble excipients and/or adjuvants that may be added are e.g.monosaccharides, disaccharides, oligosaccharides and polysaccharides,glycerol and water-soluble antioxidants. Examples of them are givenabove.

Step III

Fat-soluble active ingredients and fat-soluble colorants are those asdescribed above.

The (fat-soluble) active ingredient and/or fat-soluble colorant andoptional fat-soluble excipients and adjuvents are either used as such ordissolved or suspended in a triglyceride and/or an (organic) solvent.

Suitable organic solvents are halogenated aliphatic hydrocarbons,aliphatic ethers, aliphatic and cyclic carbonates, aliphatic esters andcyclic esters (lactones), aliphatic and cyclic ketones, aliphaticalcohols and mixtures thereof.

Examples of halogenated aliphatic hydrocarbons are mono- orpolyhalogenated linear, branched or cyclic C1- to C15-alkanes.Especially preferred examples are mono- or polychlorinated or-brominated linear, branched or cyclic C1- to C15-alkanes. Morepreferred are mono- or polychlorinated linear, branched or cyclic C1- toC15-alkanes. Most preferred are methylene chloride and chloroform.

Examples of aliphatic esters and cyclic esters (lactones) are ethylacetate, isopropyl acetate and n-butyl acetate; and γ-butyrolactone.

Examples of aliphatic and cyclic ketones are acetone, diethyl ketone andisobutyl methyl ketone; and cyclopentanone and isophorone.

Examples of cyclic carbonates are especially ethylene carbonate andpropylene carbonate and mixtures thereof.

Examples of aliphatic ethers are dialkyl ethers, where the alkyl moietyhas 1 to 4 carbon atoms. One preferred example is dimethyl ether.

Examples of aliphatic alcohols are ethanol, iso-propanol, propanol andbutanol.

Furthermore any oil (triglycerides), orange oil, limonen or the like andwater can be used as a solvent.

Fat-soluble excipients and/or adjuvants that may be added are e.g. cornoil, mono- or di-glycerides of fatty acids, polyglycerol fatty acids,and middle chain triglycerides (“MCT”).

Step IV

In an alternative process of the present invention step III) is notcarried out, but the fat-soluble active ingredient and/or fat-solublecolorant and the optional fat-soluble excipient and/or adjuvant isdirectly added to the solution of step I) or II).

Step V

For the homogenisation conventional technologies, such as high-pressurehomogenisation, high shear emulsification (rotor-stator systems),micronisation, wet milling, microchanel emulsification, membraneemulsification or ultrasonification can be applied. Other techniquesused for the preparation of compositions containing fat-soluble activeingredient(s) and/or fat-soluble colorant(s) for enrichmentfortification and/or coloration of food, beverages, animal feed,cosmetics or pharmaceutical compositions are disclosed in EP-A 0 937 412(especially paragraphs [0008], [0014], [0015], [0022] to [0028]), EP-A 1008 380 (especially paragraphs [0005], [0007], [0008], [0012], [0022],[0023] to [0039]) and in U.S. Pat. No. 6,093,348 (especially column 2,line 24 to column 3, line 32; column 3, line 48 to 65; column 4, line 53to column 6, line 60), the contents of which are incorporated herein byreference.

Step VI

The cross-linking agent is preferably selected from the group consistingof reducing sugars, glycoproteins, and glycopeptides. Thus anintermolecular cross-linking between the (modified) rice endospermprotein and the sugar or sugar part of the glycoprotein/glycopeptide isformed. Preferred examples of the cross-linking agent are themonosaccharides (fructose glucose, galactose, xylose), disaccharides(saccharose, lactose), oligosaccharides (dextrin) and polysaccharides(Xanthan gum, pectin), most preferred are fructose, glucose and Xanthangum.

Glycoprotein is a compound containing carbohydrate (or glycan)covalently linked to protein. The carbohydrate may be in the form of amonosaccharide, disaccharide, oligosaccharide, polysaccharide, or theirderivatives (e.g. sulfo- or phospho-substituted).

Preferred examples of glycoproteins are egg albumin, milk casein.

A glycopeptide is a compound consisting of carbohydrate linked to anoligopeptide composed of L- and/or D-amino acids. A glyco-amino-acid isa saccharide attached to a single amino acid by any kind of covalentbond.

A preferred example of glycopeptides is milk lactoferrin, aniron-binding glycopeptide.

Thus, in contrast to co-pending PCT/EP2006/011873 here the riceendosperm protein which is partially conjugated with mono-, di-, oligo-or polysaccharides (especially with mono- or polysaccharides) orpartially deamidated may further be partially cross-linked with at leastone compound selected from the group consisting of reducing sugars,glycoproteins or glycopeptides.

Step VIa

The cross-linking can be achieved by submitting mixtures additionallycontaining a cross-linking agent as described above to heat-treatment tocause cross-linking of the sugar with the protein in a Maillard typereaction, i.e. by thermally treatment, preferably at temperatures fromabout 30 to about 160° C., more preferably at temperatures from about 70to about 100° C., most preferably at temperatures from about 80 to about90° C.

Cross linking is an enzymatic or a non-enzymatic reaction resulting fromthe initial condensation between an available amino group of protein anda carbonyl-group of mono-, di-, oligo- or polysaccharides (especially ofmono- or polysaccharides). Cross linking is a specific type ofmodification which is being used to alter protein physicochemical andfunctional performance such as improving emulsification, encapsulation.

Further partial cross-linking of the rice endosperm protein which ispartially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidated withthe cross-linking agent can also be achieved by treatment withcross-linking enzymes (acyltransferases, EC 2.3, e.g. transglutaminase,EC 2.3.2.13, protein-glutamine:γ-glutamyltransferase), i.e. byenzymatically treatment, conveniently carried out at temperatures fromabout 0 to about 70° C., preferably at temperatures from about 20 toabout 40° C. Preferably the enzymatic treatment according to step VIa)is a treatment with a cross-linking enzyme, particularly with atransglutaminase.

Enzymatic cross-linking results in stable protein-containingpolysaccharide networks, in the case of a transglutaminase by theformation of ε-(γ-glutamyl)-lysine isopeptide bonds. The use ofglycoproteins or glycopeptides is preferred for the enzymaticcross-linking.

Both techniques, heat-treatment to cause cross-linking of the sugar withthe protein in a Maillard type reaction and enzymatic cross-linking canbe used for the incorporation of lipophilic moieties and can be carriedout either in a dried form of the composition (step IX), or in anaqueous solution or suspension (step VIa). The enzymatic cross-linkingis preferably carried out in an aqueous solution or suspension.

Step VII

The so-obtained dispersion, which is an oil-in-water dispersion, can beconverted after removal of the organic solvent (if present) into a solidcomposition, e.g. a dry powder, using any conventional technology suchas spray drying, spray drying in combination with fluidised bedgranulation (the latter technique commonly known as fluidised spraydrying or FSD), or by a powder-catch technique whereby sprayed emulsiondroplets are caught in a bed of an absorbent, such as starch, calciumsilicate and silicon dioxide, and subsequently dried.

Spray-drying may be performed at an inlet-temperature of from about 100to about 250° C., preferably of from about 150° C. to about 200° C.,more preferably of from about 160 to about 190° C., and/or at anoutlet-temperature (product temperature) of from about 45 to about 160°C., preferably of from about 55 to about 110° C., more preferably offrom about 65 to about 95° C.

Step VIII

The drying of the powder obtained in step VII is preferably carried outat a temperature of ≦100° C., preferably at a temperature of from 20 to100° C., more preferably at a temperature of from 60 to 70° C. If thedrying is performed in vacuum the temperature is lower.

Step IX

The cross-linking via heat-treatment is carried out as already describedabove for step VIa. The same applies for the enzymatic treatment, whichis, however, preferably carried out in solution/suspension.

Manufacture of the Rice Endosperm Protein which is Partially Conjugatedwith Mono-, Di-, Oligo- or Polysaccharides (Especially with Mono- orPolysaccharides) or Partially Deamidated

The present invention is also directed to a process for the manufactureof a rice endosperm protein which is partially conjugated with mono-,di-, oligo- or polysaccharides (especially with mono- orpolysaccharides) or partially deamidated starting from milled rice,whereby the rice bran was removed before milling, comprising thefollowing steps a) to e):

a) preparing an aqueous solution or suspension of milled rice, wherebythe rice bran was removed before milling, whereby the solution orsuspension preferably has a dry mass content of from 0.1 to 30 weight-%,preferably from 10 to 15 weight-%, based on the total amount of theaqueous solution or suspension;b) removing the non-protein part or the protein part of the milled rice,whereby the rice bran was removed before milling, to obtain the riceendosperm protein;c) modifying the protein part of the milled rice, whereby the rice branwas removed before milling, by reacting the protein part of the milledrice partially with mono-, di-, oligo- or polysaccharides (especiallywith mono- or polysaccharides) in a Maillard-type reaction or bypartially deamidating the protein part of the milled rice to obtain riceendosperm protein which is partially conjugated with mono-, di-, oligo-or polysaccharides (especially with mono- or polysaccharides) orpartially deamidated;d) optionally isolating the rice endosperm protein which is partiallyconjugated with mono-, di-, oligo- or polysaccharides (especially withmono- or polysaccharides) or partially deamidated;e) optionally converting the rice endosperm protein which is partiallyconjugated with mono-, di-, oligo- or polysaccharides (especially withmono- or polysaccharides) or partially deamidated into a solid form.

Step a)

Milled rice, where the rice bran was removed before milling, is alsoknown under the expression “rice flour”.

This step is simply performed by adding water to the rice flour or viceversa, optionally by stirring vigorously (with a mechanical stirrer)until the rice flour is completely dispersed, or by homogenizing therice flour suspension with a homogenizer, e.g. for 5 minutes at roomtemperature.

Step b)

Step b) may be performed as described by Paraman, I., Hettiarachchy, N.S., Schaefer, C., and Beck. M. I. in Cereal Chem. 2006, 83(6), 663-667:“Physicochemical properties of rice endosperm proteins extracted bychemical and enzymatic methods”.

Removing of the Non-Protein Part

Step b) may preferably be achieved by treating the rice flour withnon-protein degrading enzymes, e.g. with a 0.5% aqueous suspension ofTermamyl® at a temperature of 90° C. for 2 hours and then with a 0.1%aqueous suspension of a cellulase at a temperature of 50° C. for 30minutes—without any pH adjustment (pH 6-7), deactivating the enzymes,separating and removing the non-protein part from the protein part ofthe rice flour.

Preferred examples of non-protein degrading enzymes are starch-degradingenzymes such as α-amylases and cellulases, i.e. cellulose-degradingenzymes, and mixtures thereof. A preferred example of an α-amylase isTermamyl® 120, Type L, commercially available from Novo Nordisk Biochem,North America, Inc., USA. Other preferred examples are Liquzyme® Supra,commercially available from Novo Nordisk Biochem, North America, Inc.,USA, Amylase S “Amano” 35 G, commercially available from AmanoPharmaceutical Co. Ltd., Nagoya, Japan, Multifect Cellulase,commercially available from Genencor International, Inc., USA, andCellulase T “Amano” 4, commercially available from Amano PharmaceuticalCo. Ltd., Nagoya, Japan.

The reaction of the enzymes can be stopped by neutralising the solutionor suspension if an inorganic acid (e.g. hydrochloric acid) or anorganic acid (e.g. citric acid) or base is used or by heating todenature the enzymes.

The denaturation may be achieved by heating the solution to atemperature of from 80 to 95° C., preferably to a temperature of from 80to 85° C. (especially at a low pH of from 3.5 to 4.5) for 10 to 15minutes. Afterwards the solution may be cooled to 50° C.

The separation of the non-protein part may be achieved by centrifugation(5000 g for 15 minutes) (whereby the non-protein part is in the waterphase), followed by washing with deionized water. The rice endospermprotein remains in pellets.

Removing of the Protein Part

Alternatively a so-called “alkaline extraction” or a so-called“salt-extraction” may be performed before the centrifugation orfiltration.

“Alkaline extraction” means that first the pH of the solution orsuspension of the rice flour is adjusted to a value of from 7 to 12,preferably to a value of from 8 to 10, more preferably to a value ofabout 9, with an alkali solution (e.g. an aqueous NaOH solution) at 40to 60° C. for 3 hours.

In cases where the protein yield is more important than the proteinfunctionality it may be advantageous to adjust the pH preferably to avalue of from 8 to 12, more preferably of from 9 to 12, even morepreferably from 10 to 12.

Preferably such a base has a concentration of about 0.1 to 5 M,preferably of about 0.5 to about 2 M. The base may be an inorganic base.Examples of inorganic bases are (earth) alkali hydroxides such as sodiumhydroxide (preferred), potassium hydroxide and calcium hydroxide.

A “salt-extraction” is similar to an “alkali-extraction”, but inaddition to the base a salt such as sodium chloride is used. In apreferred embodiment of the invention an aqueous 0.08 M sodium chloridesolution (adjusted to pH 11 with NaOH) is used as the extractingsolvent.

In both cases (alkaline or salt extraction) the protein part istransferred to the water phase. The protein part may be separated thenby centrifugation or filtration from the non-protein part.

Further Modification of the Protein Part

A further modification of the rice flour may be achieved by treatingit(s protein part) with (commercially available) food grade alkaline,neutral and/or acid proteases. For some proteases the enzymespecifications and the optimum conditions are given in the tables below.

TABLE 1a Enzyme specification I Type of Enzyme protease SourcePreferential specificity Protex 6L Serine Protease Bacillus Hydrolysisof proteins with broad specificity licheniformis for peptide bondsBromelain Cysteine Pineapple Broad specificity, but strong preferenceProtease stem for Arg-Arg in peptides Alkalase Serine Protease BacillusBroad specificity, and a preference for a licheniformis large unchargedresidue's carboxyl sites Liquipanol Cysteine Concentrated Broadspecificity Protease papain Alkaline Serine Protease Bacterial proteaseHydrolysis of proteins with broad specificity protease for peptide bondsPepsin Aspartic Porcine stomach The C-terminal side of tyrosine,Protease phenylalanine, and tryptophan residues

TABLE 1b Enzyme specification II Enzymes pH-range Activity/g CompanyProtex 6L  6-10 580 000 Genencor International, Inc., Rochester, NY14618, USA Bromelain 5-8 150 000 Enzyme Development Corporation, NewYork, NY 10001, USA. Alkalase 6-9 2.4 AU Novo Nordisk Biochem,Franklinton, NC 27525, USA Liquipanol 5-8 125 000 Enzyme DevelopmentCorporation, New York, NY 10001, USA. Alkaline 6-9 175 000 EnzymeDevelopment Corporation, protease New York, NY 10001, USA.

TABLE 2 The optimum enzyme conditions used in protein hydrolysis Amountof Enzyme [weight-%, based on Temperature Time Enzyme protein weight] pH[° C.] [minutes] Liquipanol 1.0 8.0 50 60 Bromelain 1.0 7.0 50 60Alkalase 1.0 9.0 60 60 Protex 6L 1.0 10.0 60 60 Pepsin 0.5 3.0 37 30

The proteases may be from bacteria or fungi, as well as from fruit ormay have animal origin.

Examples of alkaline proteases are the commercially available Alkalase®(Novo Nordisk Biochem, Franklinton, N.C., USA), Alkaline Protease®(Enzyme Development Corporation, New York, N.Y., USA), Protex 6L®(Genencor® Bacterial Alkaline Protease, Genencor International, Inc.,Rochester, N.Y., USA) and Genencor® Protease 899 (GenencorInternational, Inc., Rochester, N.Y., USA).

Examples of neutral proteases are the commercially available Bromelain®(Enzyme Development Corporation, New York, N.Y., USA), Liquipanol®(Enzyme Development Corporation, New York, N.Y., USA) and bacterialneutral-protease (Genencor International, Inc., Rochester, N.Y., USA). Afurther example of a neutral protease is the commercially availableCollupilin® of DSM Food Beverages, Delft, Netherlands, produced fromCarica papaya, a plant, i.e. an enzyme of fruit origin.

Examples of acid proteases are pepsin (Sigma, USA) and Acid protease(Amano Pharmaceutical Co. Ltd., Nagoya, Japan).

In a preferred embodiment of the process of the present invention theprotein part of the rice flour is treated subsequently by two differentalkaline proteases at a pH range of from 7 to 10 for 10 to 80 minutes at40 to 60° C.

Preferably one of these proteases is a serine specific protease such asAlkalase®, Protex 6L® or Alkaline Protease® and the other is a cysteinespecific protease such as Liquipanol® or Bromelaina

This modification step may also be modified by not adding the enzyme(s)at once but by adding them (subsequently or simultaneously) portionwise.

Step c)

The protein part obtained in step b) is used as starting material forperforming step c), i.e. either a partial reaction with mono- (di- oroligo-) or polysaccharides or a partial deamidation.

Partial Reaction of the Protein Part of the Milled Rice with Mono-, Di-,Oligo- or Polysaccharides (Especially with Mono- or Polysaccharides)

The reaction of the protein part with the mono-, di-, oligo- orpolysaccharides proceeds in a Maillard-type reaction. Aqueousdispersions of the protein part and aqueous solutions/dispersions of themono-, di-, oligo- or polysaccharide (especially of the mono- orpolysaccharide), respectively, may be prepared separately and then addedtogether under stirring.

Usually the aqueous solution of the mono- or polysaccharide (or di- oroligosaccharide), most preferably having a concentration of 10 weight-%,was slowly added to the aqueous solution of the protein part, mostpreferably having a concentration of 10 weight-%, under stirring toobtain a mixture of both solutions/dispersions. The mixing is preferablycarried out at a temperature in the range of from 20 to 60° C.,preferably in the range of from 30 to 50° C., more preferably in therange of from 35 to 45° C., and for a time in the range of from 10minutes to 2 hours, preferably in the range of from 30 minutes to 90minutes, more preferably in the range of from 45 minutes to 60 minutes.

Then the pH value of the mixture is adjusted to a value in the range offrom 6.0 to 9.0, preferably to a value in the range of from 6.5 to 8.5,preferably to a value in the range of from 7.0 to 8.0 by adding a base,preferably in form of an aqueous solution. Preferably such a base has aconcentration of about 0.1 to 3 M, preferably of about 0.5 to about 2 M.

The base may be an inorganic base. Examples of inorganic bases are(earth) alkali hydroxyides such as sodium hydroxide (most preferred),potassium hydroxide (preferred) and calcium hydroxide.

The thus obtained alkaline mixture is then dried, preferably byspray-drying. The spray-drying is preferably carried out at aninlet-temperature in the range of from 50° C. to 250° C., morepreferably in the range of from 60° C. to 100° C., most preferably inthe range of from 60 to 90° C.

The reaction (cross-linking) may then be performed by incubating thedried protein and mono- or polysaccharide (or di- or oligosaccharide)mixture to a temperature in the range of from 30 to 70° C., preferablyin the range of from 40 to 60° C., more preferably in the range of from45 to 55° C. in a humidity chamber. Depending on the reactiontemperature the duration of the reaction is in a time range from 2 hoursto 60 hours, preferably from 4 hours to 40 hours, more preferably from 6hours to 30 hours. The relative humidity may vary in a range of from 39to 85%, more preferably in the range of from 44 to 79%, most preferablyin the range of from 54 to 69%, to maintain the water activity at arange of from 0.5 to 0.8.

The cross-linking may also be carried out by enzymatic treatment asalready described above for step VIa.

Preferred examples of monosaccharides are pentoses and hexoses(fructose, glucose, galactose, xylose, especially fructose and glucose).

Preferred examples of polysaccharides are Xanthan gum and pectin.

Instead of mono- or polysaccharides also disaccharides (saccharose,lactose) and oligosaccharides (dextrin) may be used.

The weight ratio of the protein part to the monosaccharide lies in therange of from 0.5 to 12% (w/w), preferably in the range of from 0.1 to8% (w,w), more preferably in the range of from 0.5 to 4% (w/w).

The weight ratio of the protein part to the polysaccharide (ordisaccharide or oligosaccharide) lies in the range of from 0.1 to 20%(w,w), preferably in the range of from 0.5 to 20% (w/w).

If glucose is used as monosaccharide the weight ratio of glucose to theprotein part obtained in step b) is preferably in the range of from 0.1to 8% (w/w), more preferably in the range of from 0.5 to 4% (w/w).

If Xanthan gum is used as polysaccharide the weight ratio of Xanthan gumto the protein part obtained in step b) is preferably in the range offrom 0.1 to 20% (w/w), more preferably in the range of from 0.5 to 10%(w/w).

Partial Deamidation of the Protein Part of the Milled Rice

The deamidation is performed by adjusting the pH value of an aqueouscolloidal solution of the protein part of the rice endosperm proteinobtained in step b) to a value in the range of from 9.0 to 13.0,preferably to a value in the range of from 9.5 to 12.5, preferably to avalue in the range of from 10.5 to 12 by adding a base, preferably inform of an aqueous solution. Preferably such a base has a concentrationof about 0.1 to 3 M, preferably of about 0.5 to about 2 M. The base maybe an inorganic base. Examples of inorganic bases are (earth) alkalihydroxides such as sodium hydroxide (most preferred), potassiumhydroxide (preferred) and calcium hydroxide.

The thus resulting alkaline mixture is then brought to a temperature inthe range of from 25 to 90° C., preferably in the range of from 30 to80° C., more preferably in the range of from 40 to 70° C.

Depending on the deamidation temperature the duration of the deamidationis in a time range from 0.5 hours to 24 hours, preferably from 0.5 to 12hours, more preferably from 0.5 to 6 hours.

Step d)

Step d) may be performed by any method known to the person skilled inthe art for isolating proteins.

Step d) may e.g. be carried out by centrifugation and/or filtration.

Usually it is not necessary to isolate the protein partially conjugatedwith mono-, di-, oligo- or polysaccharides or partially deamidated.

Step e)

The conversion into a solid form, e.g. a dry powder, can be achieved byany drying method known to the person skilled in the art. Preferred arespray drying or freeze-drying. Spray drying is preferably performed atan inlet temperature of 200° C. to 240° C. and at an outlet temperatureof 80 to 100° C. The freeze-drying is preferably performed at atemperature of from about −20° C. to about −50° C. for 10 to 48 hours.

An object of the present invention is also the rice endosperm proteinwhich is partially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidateditself, especially the one as obtainable by any process as describedabove. Even more preferred is the rice endosperm protein which ispartially conjugated with mono-, di-, oligo- or polysaccharides(especially with mono- or polysaccharides) or partially deamidateditself as obtained by any process as described above.

INDUSTRIAL APPLICABILITY

The present invention is directed to the use of a composition asdescribed above for the enrichment, fortification and/or coloration offood, beverages, animal feed, personal care or pharmaceuticalcompositions, as well as to the food, beverages, animal feed, personalcare and pharmaceutical compositions containing such a composition asdescribed above themselves.

The present invention is also directed to food, beverages, animal feed,personal care and pharmaceutical compositions containing a riceendosperm protein which is partially conjugated with mono-, di-, oligo-or polysaccharides (especially with mono- or polysaccharides) orpartially deamidated as described above, as well as to the use of such arice endosperm protein (with the preferences as described above) asprotective hydrocolloid for fat-soluble active ingredients and/orfat-soluble colorants.

Animals including humans in the context of the present inventionencompass besides humans especially farm animals such as sheep, cow,horses, poultry (broiler and egg pigmentation), shrimps and fish(especially salmon and rainbow trout) as well as pets such as cat, dogs,birds (e.g. flamingos) and fish.

Beverages wherein the compositions of the present invention can be used,especially as a colorant or a functional ingredient, can be carbonatedbeverages e.g., flavoured seltzer waters, soft drinks or mineral drinks,as well as non-carbonated beverages e.g. flavoured waters, fruit juices,fruit punches and concentrated forms of these beverages. They may bebased on natural fruit or vegetable juices or on artificial flavours.Also included are alcoholic beverages and instant beverage powders.Besides, sugar containing beverages, diet beverages with non-caloric andartificial sweeteners are also included.

Further, dairy products, obtained from natural sources or synthetic, arewithin the scope of the food products wherein the compositions of thepresent invention can be used, especially as a colorant or as afunctional ingredient. Typical examples of such products are milkdrinks, ice cream, cheese, yoghurt and the like. Milk replacing productssuch as soymilk drinks and tofu products are also comprised within thisrange of application.

Also included are sweets which contain the compositions of the presentinvention as a colorant or as a functional ingredient, such asconfectionery products, candies, gums, desserts, e.g. ice cream,jellies, puddings, instant pudding powders and the like.

Also included are cereals, snacks, cookies, pasta, soups and sauces,mayonnaise, salad dressings and the like which contain the compositionsof the present invention as a colorant or a functional ingredient.Furthermore, fruit preparations used for dairy and cereals are alsoincluded.

The final concentration of the fat-soluble active ingredient and/or thefat-soluble colorant which is added via the compositions of the presentinvention to the food products may be from 0.1 to 500 ppm, particularlyfrom 1 to 50 ppm, based on the total weight of the food composition anddepending on the particular food product to be coloured or fortified andthe intended grade of coloration or fortification.

The food compositions of this invention are preferably obtained byadding to a food product the fat-soluble active ingredient and/or thefat-soluble colorant in the form of a composition of this invention. Forcoloration or fortification of a food or a pharmaceutical product acomposition of this invention can be used according to methods per seknown for the application of water dispersible solid compositions of thepresent invention.

In general the composition may be added either as an aqueous stocksolution, a dry powder mix or a pre-blend with other suitable foodingredients according to the specific application. Mixing can be donee.g. using a dry powder blender, a low shear mixer, a high-pressurehomogeniser or a high shear mixer depending on the formulation of thefinal application. As will be readily apparent such technicalities arewithin the skill of the expert.

Pharmaceutical compositions such as tablets or capsules wherein thecompositions are used as a colorant are also within the scope of thepresent invention. The coloration of tablets can be accomplished byadding the compositions of the present invention in form of a liquid orsolid colorant composition separately to the tablet coating mixture orby adding a colorant composition to one of the components of the tabletcoating mixture. Coloured hard or soft-shell capsules can be prepared byincorporating a colorant composition in the aqueous solution of thecapsule mass.

Pharmaceutical compositions such as tablets such as chewable tablets,effervescent tablets or filmcoated tablets or capsules such as hardshell capsules wherein the compositions are used as an active ingredientare also within the scope of the present invention. The compositions ofthe present invention are typically added as powders to the tabletingmixture or filled into the capsules in a manner per se known for theproduction of capsules.

Animal feed products such as premixes of nutritional ingredients,compound feeds, milk replacers, liquid diets or feed preparationswherein the compositions are either used as a colorant for pigmentatione.g. for egg yolks, table poultry, broilers or aquatic animals(especially shrimps, salmon, rainbow trout) or as an active ingredientare also within the scope of the present invention.

Personal care compositions: Cosmetics, toiletries and derma productsi.e. skin and hair care products such as creams, lotions, baths,lipsticks, shampoos, conditioners, sprays or gels wherein thecompositions are used as a colorant or as an active ingredient are alsowithin the scope of the present invention.

The present invention is further illustrated by the following examples.

EXAMPLES

The following abbreviations are used:

DH=degree of hydrolysisDI water=deionized waterdw=dry weight basisRH=relative humidityrpm=rounds per minuteSDS=sodium dodecyl sulfatew/v=weight/volume

Rice flour made from long grain rice was provided by Riceland Foods(Stuttgart, Ark.). Whey protein isolate was obtained from Biozate®,Davisco Foods International, INC., Minnesota as benchmark standardprotein containing 88.6% protein (N×6.25) on dry weight basis. Alcalase2.4 L, bacterial serine protease from Bacillus lieheniformis, wasprovided by Novo Nordisk Biochem., (Franklinton, N.C., 2.4 AU units/g).Analytical reagents were purchased from Fisher Scientific (Pittsburgh,Pa.) and Sigma chemical Co. (St. Louis, Mo.).

(A) Analytical Methods: Examples 1-5 Example 1 Determination of EmulsionActivity and Emulsion Stability

The emulsion activity and stability was determined by the turbidimetricmethod of Pearce and Kinsella, Journal of Agric Food Chem. 1978, 26,716-722. A mixture of 6 mL of a 0.1% solution of the rice endospermprotein in 10 mM phosphate buffer of a pH of 7.0 and 2 mL of corn oilwas homogenized for 1 minute with a sonicator at setting 6 (VirtishearTempest, The Virtis Co., Gardiner, N.Y., U.S.A.). 50 microliters of themixture were transferred into 5 mL of an 0.1% aqueous solution of SDS(w/v) 0 and 10 minutes after the homogenization. The absorbance of thesolution at 500 nm was determined with a spectrometer (Shimadzu ModelUV-1601, Kyoto, Japan). The absorbance at the time 0 afterhomogenization is the emulsion activity of the rice endosperm protein.The decrease in turbidity (absorbance) of the initial absorbance duringthe time interval (10 min) was used to calculate the emulsion stability.(ES) was calculated as follows:

Emulsion Stability=To×Δt/ΔT—where, ΔT is the decrease in turbidity(absorbance) of the initial absorbance (To) during the time interval ofΔt (10 min).

Example 2 Determination of the Degree of Hydrolysis

The DH was determined by the method of Nielsen and others (Nielsen, P.M., Petersen, D. & Dambmann, C.: Improved method for determining foodprotein degree of hydrolysis. Journal of Food Science 2001, 66 (5),642-646). The o-phthaldialdehyde (OPA) reagent was prepared as follow:7.620 g of di-sodium tetraborate decahydrate (Na₂B₄O₇.10H₂O) and 200 mgsodium dodecyl sulfate (SDS) were dissolved in 150 mL of deionized waterand then mixed with 160 mg of OPA (97% OPA pre-dissolved in 4 mL ofethanol) and 176 mg of 99% dithiothreitol (DTT). The final solution wasmade up to 200 mL with deionized water. Freeze dried protein sample of0.1 g was solubilized in 10 mL deionized water. To measure theabsorbance, 3 mL of OPA reagents was added to 10 mL tubes and then 400μL of sample solution, serine standard (10 mg/l 00 mL) and deionizedwater was added in four tubes for each sample, standard and blank,respectively. This was followed by mixing for 5 s and held for exactly 2min. Absorbance was read at 340 nm with a spectrophotometer (ShimadzuModel UV-1601, Kyoto, Japan). The DH was calculated as follows.

DH=h/h_(total)*100%; where h is the number of hydrolyzed bonds andh_(total) is the total number of peptide bonds per protein equivalent;h=(Serine-NH2-β)/α equiv/g protein; where h is the number of hydrolyzedbonds and h_(total) is the total number of peptide bonds per proteinequivalent; for cereal protein α is 1.00, β is 0.40, and h_(total) is8.0.

Serine-NH2=[(A_(340sample)−A_(340blank))/(A_(340standard)−A_(340blank))]*0.9516meqv/L*0.01*100/(X*P); where serine-NH2=meqv serine NH2/g protein; X=gsample; P=% protein in sample; 0.01 is the sample volume in liter (L).

Example 3 Determination of the Protein and Total Solubility

Protein solubility (also N-solubility) was determined by the method ofBera and Mukherjee (Bera, M. B., Mukherjee, R. K.: Solubility,emulsifying, and foaming properties of rice bran protein concentrates. JFood Sci 1989, 54(1), 142-145) with some modifications. 200 mg ofprotein sample was dispersed in 10 mL of deionized water, the pH wasadjusted to 7.0 by 1 N HCl or 1 N NaOH. The dispersion was stirredcontinuously for 30 min and centrifuged at 5000 rpm for 15 min. (modelJ2-21, Beckman, Fullerton, Calif., U.S.A.). The supernatant wasrecovered, and the protein content in the supernatant was determined bythe Automatic Kjeldahl method (AACC 1990). The percentage of proteinsolubility was calculated by following equation:

${{Protein}\mspace{20mu} {Solubility}\mspace{14mu} (\%)} = {\frac{{Protein}\mspace{14mu} {content}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {supernatant}}{{{Protein}\mspace{14mu} {in}\mspace{14mu} 200\mspace{14mu} {mg}\mspace{14mu} {protein}} - {isolate}} \times 100}$

The protein solubility was calculated as the percent ratio of protein inthe supernatant to that of the total protein in the initial sample.

The total solubility was determined by oven drying method, and expressedas the percent ratio of total soluble portion of the supernatant to thatof the total weight of the protein isolate.

${{Total}\mspace{14mu} {Solubility}\mspace{14mu} (\%)} = {\frac{{Soluble}\mspace{14mu} {portion}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {supernatant}}{{200\mspace{14mu} {mg}\mspace{14mu} {protein}} - {isolate}} \times 100}$

Example 4 Determination of Viscosity

Viscosity of the protein isolates was determined by a rotationalrheometer (Haake VT 550, Germany) equipped with a MVDIN measuringspindle (radius=19.36 mm, height=58.08 mm) at room temperature (26° C.).The protein isolates were mixed with deionized water to form slurry of10%, and the slurry was left for 60 min for equilibrium before analysis.The samples (30 ml) were loaded into the cylindrical cup (radius=21.0mm) and were subjected to a shear rate that changed from 0 to 400 l/sover 3 min using a computer-controlled program. The data were analyzedby Rheowin Pro Data manager version 2.84 (Haake Mess Tech, Germany).

Example 5 Determination of the Protein Content

The protein contents were determined by an Automatic Kjeldahl method(AACC, 1990). The Kjeldahl 2006 Digester (Foss Tecator, Hoganas, Sweden)was used for digesting the samples in concentrated sulfuric acid withKjeldahl® tablet as catalyst at 420° C. for 1 hour, and a Kjeltec® 2300Analyzer Unit (Foss Tecator, Hoganas, Sweden) was used to determine thenitrogen content of the products. The protein contents wereautomatically calculated by multiplying the nitrogen content by a factorof 5.95.

(B) Manufacture of Rice Endosperm Protein Isolates (RP) (StartingMaterial and Comparison Example) Example 6

One kilogram of rice flour was homogenized with 8 L deionized water(1:8, w/v) in a homogenizer (Virtishear Tempest, The Virtis Co.,Gardiner, N.Y., USA) for 1 minute. The pH of the slurry was adjusted to11.0 by 1M NaOH, and the suspension was stirred for 3 hours at 40° C.The soluble protein in the solution was separated by centrifugation(5,000 rpm, 15 minutes). This procedure was repeated once to extractadditional protein from the residue. Proteins in the combinedsupernatants of first and second extractions were isoelectricallyprecipitated at pH 4.5 and kept at 4° C. for 1 hour. The precipitate wasrecovered by centrifugation at 5,000 rpm for 20 minutes, washed withdeionized water (1:4, w/v, pH 4.5), adjusted to pH 7.0. The extractedproteins (RP) were used as a starting material for the preparation ofthe following modifications (C) and (D).

Experimental Design of Examples (C), (D) and (E)

The present study includes two sets of experiments: (1) optimizationrice protein glycosylation and, (2) comparison of controlledglycosylation, deamidation, and alcalase modification methods(comparison examples) on rice protein physicochemical properties. Theoptimization of glycosylation experiment was conducted in a 2×7 twofactor factorial design (glucose and Xanthan gum) with repeatedmeasurements (7 reaction-times) with 3 replicates. The comparison ofglycosylation, deamidation, and alcalase modification methods wereconducted in one factor completely randomized design, which includedcontrol (unmodified) rice protein and bench mark whey protein toevaluate and compare the effectiveness of the selected modificationmethods.

Thus, the rice endosperm protein was modified by the following methods:(1) Glycosylation of rice endosperm protein with D-glucose (RP_(Glu));(2) Glycosylation of rice endosperm protein with Xanthan gum (RP_(XG));(3) Deamidation of rice endosperm protein using alkali treatment(RP_(DA)); (4) Treatment of rice endosperm protein with alcalase to 1.8%DH (RP_(Alc)). The physicochemical and functional properties of theprotein derivatives were evaluated and compared with those of unmodifiedrice endosperm protein (RP) and as bench mark compared with whey proteinisolates.

(C) Manufacture of a Partially Hydrolyzed Rice Endosperm Protein Example7 (Comparison Example) Treatment of Rice Endosperm Protein with Alcalaseto 1.8% DH(RP_(Alc))

The conditions for alcalase treatment were chosen based on a previousstudy (Paraman et al 2007) and our preliminary data. The rice endospermprotein isolate (RP) was homogenized with DI water (8% w/v) and,adjusted to pH 6.5. The protein colloidal solution was treated with 0.1%alcalase at 40° C. for 8.5 minutes. The enzyme was inactivated at 85° C.for 7 minutes. The hydrolysate was cooled immediately to 30° C. byadding ice, and the pH was readjusted to 7.0. The protein hydrolysatewas spray dried and stored at 5° C. in air tight containers until theywere used (RP_(Alc)).

(D) Manufacture of a Rice Endosperm Protein Partially Conjugated withMono- or Polysaccharides

Example 8 Evaluation of Optimal Reaction Conditions for the PartialGlycosylation of Rice Endosperm Protein

The extracted rice protein RP (20 g on dw) was dissolved in deionizedwater to give a 10% (w/v) protein colloidal solution. D-glucose (0.465g) dissolved in 10 mL DI water was added into the protein solution whilestirring the protein solution at 37° C. The protein-glucose mixture wasadjusted to pH 8.0 and stirred for 1 h at 37° C. The mixture wasfreeze-dried and the dried protein-sugar mixture was placed in aluminumplate, and incubated at 50° C. in an incubator maintained at 65%relative humidity. Approximately, 2.5 g of the protein-glucose mixturewere drawn as samples at 4 hours intervals for 24 hours. Theglycosylated protein was stored at 5° C. in air tight containers untilthey were used.

Rice protein-Xanthan gum optimization was conducted essentially similarto the method of protein-glucose optimization described above except forthe following changes; the protein-Xanthan gum ration was 100:1, and thepH of the protein-Xanthan gum solution was adjusted to a value of 7.0.

Example 9 Glycosylation of Rice Endosperm Protein with D-Glucose(RP_(Glu))

Glycosylation was conducted based on above optimization and theconditions chosen from literature (Kato at al 1990, Kato at al 1991,Achouri et al 2005, Oliver et al 2006) with some modifications asdescribed below. The alkali extracted rice protein isolates RP (200 g ondw) were dissolved in water to give a 10% (w/v) protein colloidalsolution. D-glucose (4.65 g) dissolved in 100 mL DI water was added tothe protein solution while stirring the protein solution at 37° C. Theprotein-glucose mixture was adjusted to pH 8.0 and stirred for 1 hour at37° C. The mixture was spray-dried and stored at 5° C. in air tightcontainers until they were used. The spray-dried protein-sugar mixturewas placed in aluminum plate and incubated for 8 hours at 50° C. and 65%RH. The glycosylated protein (RPGlu) was stored at 5° C. until its use.

Example 10 Glycosylation of Rice Endosperm Protein with Xanthan Gum(RP_(XG))

The rice endosperm protein isolate (RP) 200 g, on dw basis, wasdissolved in water to give a 10% (w/v) protein colloidal solution.Xanthan gum (2 g) was dissolved separately in water to give 1% (w/v)xanthan-gum solution. The xanthan gum solution was added into theprotein solution while stirring the protein solution at 37° C. Theprotein-xanthan gum mixture was spray-dried, and stored at 5° C. in airtight containers until they were used. The spray-dried protein-Xanthangum mixture was placed in aluminum plate and incubated for 20 hours at50° C. and 65% RH. The glycosylated protein (RP_(XG)) was stored at 5°C. until analyzed.

Example 11 Glycosylation of Rice Endosperm Protein with Potato Dextrin(RP_(PD))

The rice endosperm protein isolate (RP) 200 g, on dw basis, wasdissolved in water to give a 10% (w/v) protein colloidal solution.Potato dextrin (2 g) was dissolved separately in water to give 1% (w/v)potato dextrin solution. The potato dextrin solution was added into theprotein solution while stirring the protein solution at 37° C. Theprotein-dextrin mixture was adjusted to pH 7.0, stirred for 1 hour at37° C., spray-dried, and stored at 5° C. in air tight containers untilthey were used. The spray-dried protein-dextrin mixture was placed inaluminum plate and incubated for 20 hours at 50° C. and 65% RH. Theglycosylated protein (RP_(PD)) was stored at 5° C. until analyzed.

Example 12 Glycosylation of Rice Endosperm Protein with Cyclodextrin(RP_(CD))

The rice endosperm protein isolate (RP) 200 g, on dw basis, wasdissolved in water to give a 10% (w/v) protein colloidal solution.Cyclodextrin (2 g) was dissolved separately in water to give 1% (w/v)cyclodextrin solution. The cyclodextrin solution was added into theprotein solution while stirring the protein solution at 37° C. Theprotein-cyclodextrin mixture was adjusted to pH 7.0, stirred for 1 hourat 37° C., spray-dried, and stored at 5° C. in air tight containersuntil they were used. The spray-dried protein-cyclodextrin mixture wasplaced in aluminum plate and incubated for 20 hours at 50° C. and 65%RH. The glycosylated protein (RP_(CD)) was stored at 5° C. untilanalyzed.

Example 13 Glycosylation of Rice Endosperm Protein with Pectin (RP_(P))

The rice endosperm protein isolate (RP) 200 g, on dw basis, wasdissolved in water to give a 10% (w/v) protein colloidal solution.Pectin (2 g) was dissolved separately in water to give 1% (w/v) pectinsolution. The pectin solution was added into the protein solution whilestirring the protein solution at 37° C. The protein-pectin mixture wasadjusted to pH 7.0, stirred for 1 hour at 37° C., spray-dried, andstored at 5° C. in air tight containers until they were used. Thespray-dried protein-pectin mixture was placed in aluminum plate andincubated for 20 hours at 50° C. and 65% RH. The glycosylated protein(RP_(P)) was stored at 5° C. until analyzed.

(E) Manufacture of a Partially Deamidated Rice Endosperm Protein Example14 Deamidation of Rice Endosperm Protein Using Alkali Treatment(RP_(DA))

The deamidation conditions were chosen based on literature information(Schwenke 1997, Cabra et al 2007) with some modifications as describedbelow. Alkali extracted rice protein isolate was mixed with DI water (8%w/v). The protein colloidal solution was adjusted to pH 11.0 and stirredat 25° C. for 12 hours. Then, the temperature of the protein solutionwas increased under stirring for 30 min at 70° C. The solution wascooled immediately to 30° C. by adding ice, and the pH was readjusted to7.0. The deamidated protein solution was spray dried, and stored at 5°C. (RP_(DA)).

(F) Results Statistical Analysis

All the experiments were conducted in duplicate. Data were analyzed forvariance and multiple mean comparisons with JMP 6 software (SAS Inst2002). The significance of difference between means was determined bythe Tukey HSD procedure at the 5% significance level (P<0.05).

Optimization of the Glycosylation of Rice Endosperm Protein

The glycosylation of rice protein with D-glucose and Xanthan gum wereoptimized as described above. The solubility and emulsifying propertiesof the glycosylated proteins are presented in Table 3.

TABLE 3 Solubility and emulsifying properties of D-glucose and Xanthangum glycosylated rice protein as a function of incubation time at 50° C.and 65% relative humidity Glycosylation D-glucose glycosylated Xanthangum glycosylated treatments: rice protein rice protein Reaction EmulsionEmulsion Emulsion Emulsion time (h = Solubility activity stabilitySolubility activity stability hour(s)) (%) (A₅₀₀) (min) (%) (A₅₀₀) (min)RP-control 18.0 0.266 14.7 18.0 0.266 14.7 RP-0 h 23.6 0.371 15.4 26.10.479 18.1 RP-4 h 28.9 0.707 20.4 27.0 0.497 20.8 RP-8 h 34.4 0.712 23.430.0 0.515 24.5 RP-12 h 33.5 0.627 25.4 32.3 0.523 24.0 RP-16 h 29.70.601 26.8 33.1 0.583 25.5 RP-20 h 28.3 0.462 27.2 31.8 0.629 26.4 RP-24h 24.3 0.350 28.0 33.0 0.632 27.0

Glycosylation of rice protein D-glucose (2.25%, w/w) and riceprotein-Xanthan gum (1%, w/w) conjugates were prepared at 50° C. and 65%relative humidity for varying incubation time (0-24 hours). Since thereaction starts very fast, even at the beginning a difference comparedto untreated rice endosperm protein isolate was recognized.

The functional properties of the glycosylated proteins differedsignificantly as a function of Maillard reaction time and depended onthe type of sugar used to glycosylate the protein. For D-glucose, theoptimum Maillard reaction time was 8 hours at 50° C. and 65% RH. Theglycosylated proteins demonstrated a gradual improvement in solubilityand emulsifying properties as a function of Maillard reaction up to 8hours. This trend was illustrated by an increase in solubility from 23%to 34%, and the emulsion activity from 0.371 to 0.712 as reaction timeprogressed from 0 hours to 8 hours. These properties decreased beyond 8hours of incubation. However, the emulsion stability of the glycosylatedproteins gradually increased from 15.4 to 28 minutes, as the function ofincubation time from 0 to 24 hours. The increment of emulsion stabilitywas higher at the early stage of incubation (0-12 hours) than that ofthe late stage (12-24 hours), which might be due to decreasingavailability of amino groups with progressing Maillard reaction.

In Xanthan gum glycosylated proteins, the solubility and emulsifyingproperties gradually increased up to 16 and 20 hours, respectively. Thesolubility increased from 26% to 33% in 16 hours incubation; theemulsifying activity increased from 0.479 to 0.629 in 20 hours ofincubation. Compared to glucose, Xanthan gum mediated glycosylationimproved the functional properties at a slower rate and required moretime for Maillard reaction. A 18-20 hour period of incubation was theoptimum reaction time for Xanthan gum at 50° C., 65% RH, and 1:100Xanthan gum to protein ratio. The optimum reaction time varied anddepended on the reactants and reaction conditions (Oliver 2006). Simplesugars can react to faster and required shorter duration thanpolysaccharides.

In the present study, the optimal reaction times were much shorter thanthe studies published previously. Lysozyme glycosylated with Xanthan gumdemonstrated superior emulsifying properties after 7 days of Maillardreaction (Nakamura et al 2000). Similarly, Ovalbumin conjugated withglucose reached maximum emulsion activity in 1 day and emulsionstability in 14 days of reaction time (Kato et al 1995). The differencesin time requirement might be due to the differences in pre-incubationconditions used in these experiments. For instance, in these twoprevious studies, protein-sugar mixture was adjusted to pH 7.0 andimmediately freeze-dried. However, in the present study, theprotein-sugar mixture was stirred for 1 h before drying. The pH of thesugar-rice protein mixture was maintained at pH 8.0. The slight alkalicondition (pH 8.0) and longer mixing condition (1 hour) might havefacilitated Maillard reaction in the liquid stage.

The solubility and emulsifying data of native and glycosylated proteinwith 0 h incubation supported this presumption (Table 3). The solubilityand emulsifying properties of glycosylated protein with 0 hourincubation showed higher solubility and emulsifying properties than thatof native rice protein controls. For glucose-rice protein conjugate, thesolubility improved from 18 to 24% and emulsion activity improved from0.266 to 0.371 without any dry-stage reaction (Table 3); for Xanthangum-rice protein conjugate, the solubility increased from 18 to 26%,emulsion activity and stability increased from 0.266 to 0.479 and from14.7 to 18.1 min, respectively.

In Glycosylation Cross-Linking Study, Preliminary Experiments wereConducted with D-Glucose, Potato-Dextrin, Cyclodextrin, Pectin, XanthanGum.

Based on our preliminary data presented in (Table 4), D-glucose andXanthan gum conjugation significantly improved both solubility andemulsifying properties compared to rice endosperm protein andpotato-dextrin, cyclodextrin, or pectin conjugated rice protein. Thus,D-glucose and Xanthan gum were selected for further process optimizationof glycosylation.

Potato-dextrin, cyclodextrin conjugation did not improve the endospermprotein solubility, but slightly improved the emulsifying propertiescompared to control rice endosperm protein. Pectin conjugation did notimprove the solubility or emulsifying properties.

TABLE 4 Solubility and emulsifying properties of rice endosperm proteinglycosylated with various carbohydrates at 50° C. and 65% relativehumidity for 12 and 24 hours Incubation Emulsion Emulsion Type of timeactivity stability carbohydrate (h) Solubility (%) (A₅₀₀) (min) Controlrice protein 17.4 0.259 15.8 D-glucose 12 31.4 0.596 23.1 D-glucose 2426.7 0.417 26.2 Potato-dextrin 12 16.1 0.367 18.6 Potato-dextrin 24 17.90.339 20.4 Cyclodextrin 12 14.8 0.317 24.3 Cyclodextrin 24 11.8 0.29422.5 Pectin 12 14.1 0.266 15.8 Pectin 24 13.7 0.386 17.9 Xanthan gum 1229.7 0.509 22.6 Xanthan gum 24 35.7 0.577 28.3

The protein:carbohydrate ratios used in this experiment were 100:2.265for glucose, and 100:1 for potato-dextrin, cyclodextrin, pectin, andXanthan gum.

The optimized glycosylated rice proteins were further compared andevaluated for physicochemical and functional properties of the proteinsmodified by controlled enzymatic hydrolysis and alkali deamidation. Thetreatments and brief methods of modification are summarized in Table 5.

TABLE 5 Summary of rice protein modification methods Rice proteinisolate Brief out line type of rice endosperm protein preparationRP_(Control) RP Rice endosperm protein with no further modificationRP_(Alcalase) RP_(Alc) Alcalase treated rice endosperm protein to 1.8%DH RP_(Glucose) RP_(Glu) Glycosylated rice endosperm protein withD-Glucose RP_(Xanthan Gum) RP_(XG) Glycosylated rice endosperm proteinwith Xanthan gum RP_(Deamidation) RP_(DA) Alkali deamidated riceendosperm protein

The rice endosperm protein was modified by the above methods: (1)Glycosylation of rice endosperm protein with D-glucose (RP_(Glu)); (2)Glycosylation of rice endosperm protein with Xanthan gum (RP_(XG)); (3)Deamidation of rice endosperm protein using alkali treatment (RP_(DA));(4) Treatment of rice endosperm protein with alcalase to 1.8%DH(RP_(Alc)) to improve the solubility and emulsifying properties

Influence of Controlled Glycosylation, Deamidation, and AlcalaseModifications on Rice Protein Physicochemical and Functional PropertiesPhysicochemical Properties

Protein and moisture content of the rice protein products are presentedin Table 6.

TABLE 6 Moisture, protein content, and viscosity of rice proteinisolates modified by control enzymatic hydrolysis, glycosylation anddeamidation Protein Protein (%) Rice protein pH (as Moisture (as (Dryweight Viscosity isolate type is) % is, %) basis) (mPas)* RP_(Control)7.1 2.7 84.6 86.7 8.7 RP_(Alcalase) 7.4 3.3 83.7 86.5 7.0 RP_(Glucose)6.9 8.8 77.4 84.9 12.4 RP_(Xanthan Gum) 6.8 9.0 75.9 83.4 31.6RP_(Deamidation) 6.9 4.5 74.2 77.6 14.0 Whey protein 8.4 3.9 85.2 88.65.6 Values are means of duplicates and expressed on dry weight basis.Mean values with different letters in the same column are significantlydifferent (P < 0.05). *Viscosity at 26° C., at constant shear rate of400 s⁻¹; 1 Pas (Pascal second) = 1000 cP (centipoises = mPas); Viscositywas measured on ‘as is’ basis.

The glycosylated proteins, RP_(Glu), RP_(XG), contained higher moisturecontent (8.8-9.0%) than that of other proteins. These two products,RP_(Glu), RP_(XG), absorbed moisture during the glycosylation processthat was carried out at 65% RH for 8 and 18 hours, respectively. Proteincontent of the glycosylated proteins (RP_(Glu), RP_(XG)) did not differfrom the control rice protein isolates (RP) as low percent of glucose(2.25%, w/w) and Xanthan gum (1%, w/w) were used to glycosylate theproteins.

The viscosity of the protein proteins are presented in Table 6 and FIG.1.

FIG. 1 shows the relationship between shear stress (y-axis; in Pa) andshear rate α-axis; in sec⁻¹) for rice protein isolates (—RP_(control)),hydrolysate (♦P_(Alc)), glycosylated (◯RP_(Glu) and ▪RP_(XG)), anddeamidated (▴RP_(DA)) rice protein at 10% w/v concentration; 1 Pas(Pascal second)=1000 cP (centipoises=mPas). The viscosity was the slopeof shear stress and shear rate of the linear line.

Glycosylated (RP_(Glu), RP_(XG)) and deamidated (RP_(DA)) proteinsshowed much higher viscosity than the protease treated (RP_(Alc)) andcontrol rice proteins (RP). The increased viscosity can be an indicationof increasing hydrophilicity of the glycosylated. Particularly,glycosylation with Xanthan gum increased the viscosity of the product to31.6 mPas, which was significantly higher than that of glucose mediatedglycosylation (12.4 mPas) and deamidation (14.0 mPas) methods. Thehigher viscosity of Xanthan gum conjugated protein might be due thecomplex size of the glycosyl residue. In general, increasing viscosityof glycosylated proteins indicated the structural changes in protein.Glycosylated proteins have greater hydrodynamic volume and increaseshydration properties due to the increase of surface hydrophilicity andpartial unfolding of quaternary structure of protein (Baniel et al1992).

to The deamidation also increased the viscosity from 8.7 to 14.0 mPas.This indicated the improved hydration properties of the deamidatedproteins. Deamidation of amide groups into carboxyl groups mightpossibly alter the charge content of the protein and improved theinteraction of protein with water molecules. However, considering thecondition use to perform the deamidation, it is obvious that theviscosity of the deamidated protein was the net result of deamidation,denaturation, and peptide bond cleavage.

The alcalase treatment reduced the viscosity slightly from 8.7 to 7.0mPas in RP_(Alc). The reduction in viscosity was due to the reduction ofprotein molecular size. However, the viscosity change was small, sincethe protein hydrolysis was controlled to 1.8% DH.

Protein Solubility

The solubility and emulsifying properties of the proteins are presentedin Table 7.

TABLE 7 Nitrogen solubility and emulsifying properties of the riceprotein isolates Degree Emulsion Emulsion Rice protein of hydrolysis Nsolubility activity stability isolates (DH)* (%) (A500) (min)RP_(Control) — 18.0 0.266 14.7 RP_(Alcalase) 1.8 33.3 0.468 17.5RP_(Glucose) — 39.7 0.721 26.8 RP_(Xanthan Gum) — 38.6 0.661 26.1RP_(Deamidation) — 68.3 0.776 24.0 Whey protein — 97.2 0.952 18.8 Valuesare means of duplicates and expressed on dry weight basis. Mean valueswith different letters in the same column are significantly different (P< 0.05). *DH values are presented only with the samples that weresubjected to protease treatments.

Glycosylation with glucose and Xanthan gum increased the solubility ofrice protein to ˜40%, however, the increment of solubility byglycosylation was not substantial to the level expected.

Alcalase treatment (1.8%, DH) improved the solubility from 18% to 33%.The increasing solubility was due to decreasing molecular size andincreasing number of polar groups (Nielsen 1997). A previous studyreported that a high percent of the rice protein remained insoluble evenafter 13.5% DH by alcalase enzyme (Paraman et al 2007), which suggestedthat the either peptides cleaved by proteases were associated withunhydrolyzed protein via inter molecular hydrophobic or sulfhydrylinteractions or the amorphous regions of the protein only accessible tohydrolysis.

The deamidated protein showed higher solubility (68%) than any othertreatments (28-39%). The deamidated protein showed 68.3% solubility. Thedeamidation in alkali condition might have deamidated the side chainamide group of the aspartic and glutamic amino acids and possiblyalerted the protein polarity. Deamidation increase the negative chargeand can disrupt hydrophobic and hydrogen bonds (Schwenke 1997). Thesestructural changes can contribute to an increase in solubility.Particularly, the most abundant amino acids in rice glutelin areglutamine, asparagine, arginine, glycine, and alanine (Wen and Luthe1985). Deamidation of these amino acid residues can facilitate proteinsolubility, and contributed to an improvement in emulsifying properties.Further, partial cleavage of peptide bonds could be possible in thedeamidation process. The combined effect of hydrolysis along withdeamidation might have contributed to the great improvement ofsolubility.

Emulsifying Properties

The emulsion activity and stability of the glycosylated and deamidatedproteins were significantly higher than that of protease treated riceprotein isolates. The glycosylated protein with glucose showed 0.721emulsion activity and 26.8 min emulsion stability. The deamidatedprotein showed 0.776 emulsion activity and 24.0 min emulsion stability.The emulsifying properties of these two protein isolates are higher thanthat of unmodified and protease modified rice protein isolates.Glycosylation makes the proteins more hydrophilic (Kato et at 1991).Therefore, controlled and limited glycation improves emulsifyingproperties. Increasing hydrophilic nature improves the hydration andsolubility and thus possibly opens up the globular structure of protein.The partial unfolding could have improved the ability of theglycosylated protein to form stable interfacial film at the oil-waterinterface.

In the case of deamidated protein, improvement in emulsion propertiesmay be attributed by increasing protein solubility and polarity(Schwenke 1997). In addition to deamidation, as discussed earlier, thepartial cleavage of peptide bond also might have contributed to theimprovement of solubility and emulsifying properties. Increasedsolubility of the deamidated protein might help produce stableinteraction at the oil-water interface and thus improved the emulsifyingproperties. The proper balances of deamidation and peptide bond cleavagemight be the key in improving the emulsion properties rice protein bydeamidation.

The alcalase treatment to 1.8% DH(RP_(Alc)) improved emulsion activityfrom 0.266 to 0.468, and emulsion stability from 17.7 to 17.5 mincompared to unmodified rice protein (RP). In general, a low degree ofhydrolysis is recommended to improve the functionality of food proteins,especially emulsion stability. Limited proteolysis could improvemolecular flexibility and hydrophobic-hydrophilic balance of proteinwhich resulting better emulsification (Nielsen 1997; Schwenke 1997).However, the limited improvement of solubility and emulsifyingproperties might be due to (1) high hydrophobic and sulfhydrylinteractions, and exposure of buried hydrophobic regions of protein thataccompanied high temperature enzyme inactivation may have promotedaggregation and cross linking of partially hydrolyzed proteins (Paramanet al 2007). Further, due to the compact nature of the protein, theenzyme alcalase might have a limited access to interior peptide bonds,which could possibly lead to the formation of uneven size of peptide anddecrease the emulsifying properties of the resulting hydrolysate.

CONCLUSION

To improve the solubility and emulsifying properties of rice endospermprotein, glycosylation and deamidation type modifications were moreeffective than proteolysis modification by alcalase to 1.8% DN. In riceendosperm protein glycosylation, the optimum Maillard reaction time was8 h and 20 H at 50° C. and 65% RH for D-glucose and Xanthan gum,respectively. The deamidated protein showed the highest solubility (68%)and emulsifying properties (0.776-emulsion activity, 24 min emulsionstability) among the proteins evaluated in this study. The controlledenzymatic hydrolysis by alcalase 1.8% DH only improved emulsion activityfrom 0.266 to 0.468, and emulsion stability from 14.7 to 17.5 mincompared to unmodified rice protein (RP). The highest solubility andemulsifying properties of deamidated protein were the net result ofdeamidation, peptide bond cleavage, and protein denaturation that tookplace in the process of deamidation.

(G) Manufacture of a Composition of a Fat-Soluble Active Ingredient or aFat-Soluble Colorant According to the Present Invention Example 15Manufacture of a Formulation of Vitamin E (Acetate)

A formulation comprising a rice endosperm protein and vitamin E may beprepared as follows:

a) Preparation of an Emulsion:

20 g of RP_(Glu) (Glycosylation of rice endosperm protein withD-glucose) according to Example 9 were mixed with 60 g of sucrose andre-dissolved in 140 ml of water by stirring at 75° C. for 15 minutes.88.3 g of dl-α tocopherol acetate were heated to 70° C. and undervigorous stirring added to the aqueous solution. The dispersion wasvigorously stirred for another 15 minutes. Under gently stirring further225 mL of water were added and the so-obtained emulsion wascharacterised with respect to the particle size of the inner phase. Themean particle size (Sauter diameter, D[3, 2]) of the inner phase of theemulsion was 1.86 am as measured by laser diffraction (MalvernMasersizer). After storage for 12 hours, the emulsion was againcharacterised with respect to the particle size of the inner phase. Themean particle size (Sauter diameter, D[3, 2]) of the inner phase of theemulsion after storage was 1.81 μm as measured by laser diffraction(Malvern Masersizer).

b) Preparation of a Solid Formulation from the Emulsion:

The emulsion may be sprayed into a pre-cooled fluidised bed ofcornstarch. Excess cornstarch can be removed by sieving and the powderobtained can be dried in an air stream at room temperature. The powderparticle fraction in the range of 0.16 to 0.63 mm can be collected bysieving Alternatively, the emulsion can be converted into a solid formby using further well known drying technologies such spray drying.

Example 16 Manufacture of a Formulation of Vitamin E (Acetate)

A formulation comprising a rice endosperm protein and vitamin E may beprepared as follows:

a) Preparation of an Emulsion:

20 g of RP_(XG) (Glycosylation of rice endosperm protein with Xanthangum) according to Example 10 were mixed with 60 g of sucrose andre-dissolved in 140 ml of water by stirring at 75° C. for 15 minutes.83.2 g of dl-α tocopherol acetate were heated to 70° C. and undervigorous stirring added to the aqueous solution. The dispersion wasvigorously stirred for another 15 minutes. Under gently stirring further225 mL of water were added and the so-obtained emulsion wascharacterised with respect to the particle size of the inner phase. Themean particle size (Sauter diameter, D[3, 2]) of the inner phase of theemulsion was 1.65 am as measured by laser diffraction (MalvernMasersizer). After storage for 12 hours, the emulsion was againcharacterised with respect to the particle size of the inner phase. Themean particle size (Sauter diameter, D[3, 2]) of the inner phase of theemulsion after storage was 1.64 μm as measured by laser diffraction(Malvern Masersizer).

b) Preparation of a Solid Formulation from the Emulsion:

The emulsion may be sprayed into a pre-cooled fluidised bed ofcornstarch. Excess cornstarch can be removed by sieving and the powderobtained can be dried in an air stream at room temperature. The powderparticle fraction in the range of 0.16 to 0.63 mm can be collected bysieving alternatively; the emulsion can be converted into a solid formby using further well known drying technologies such spray drying.

Example 17 Manufacture of a Formulation of Vitamin E (Acetate)

A formulation comprising a rice endosperm protein and vitamin E may beprepared as follows:

a) Preparation of an Emulsion:

20 g of RP_(DA) (Deamidation of rice endosperm protein using alkalitreatment) according to Example 14 were mixed with 60 g of sucrose andre-dissolved in 140 ml of water by stirring at 75° C. for 15 minutes.81.9 g of dl-α tocopherol acetate were heated to 70° C. and undervigorous stirring added to the aqueous solution. The dispersion wasvigorously stirred for another 15 minutes. Under gently stirring further225 mL of water were added and the so-obtained emulsion wascharacterised with respect to the particle size of the inner phase. Themean particle size (Sauter diameter, D[3, 2]) of the inner phase of theemulsion was 1.77 μm as measured by laser diffraction (MalvernMasersizer). After storage for 12 hours, the emulsion was againcharacterised with respect to the particle size of the inner phase. Themean particle size (Sauter diameter, D[3, 2]) of the inner phase of theemulsion after storage was 1.68 μm as measured by laser diffraction(Malvern Masersizer).

b) Preparation of a Solid Formulation from the Emulsion:

The emulsion may be sprayed into a pre-cooled fluidised bed ofcornstarch. Excess cornstarch can be removed by sieving and the powderobtained can be dried in an air stream at room temperature. The powderparticle fraction in the range of 0.16 to 0.63 mm can be collected bysieving Alternatively, the emulsion can be converted into a solid formby using further well known drying technologies such spray drying.

Example 18 Manufacture of a Formulation of β-Carotene

A formulation comprising a rice endosperm protein and β-carotene may beprepared as follows:

a) Preparation of a(n Oil-Based) Solution 1:

6.6 g of corn oil and 1.2 g of dl-α-tocopherol were mixed. 13.8 g ofcrystalline β-carotene were dispersed in 180 ml of chloroform(trichloromethane) and the resulting dispersion to was added to themixture of corn oil and tocopherol. By gently stirring and simultaneousheating the mixture to about 60° C. a solution was obtained.

b) Preparation of a(n Aqueous) Solution 2:

30 g of RP_(Glu) (Glycosylation of rice endosperm protein withD-glucose) according to Example 9 was re-dissolved in 120 ml of water bystirring at 60° C.

c) Preparation of an Emulsion from the Solutions 1 and 2:

Under vigorous stirring solution 1 was added to solution 2 at 53° C. andthe dispersion was vigorously stirred for another 30 minutes. Thestirred dispersion was kept at 50 to 55° C. for 30 minutes. Residualtrichloromethane was removed at 50 to 55° C. After removing entrappedair bubbles by centrifugation the emulsion was gently stirred at 50 to55° C. for some minutes and then characterised with respect to theparticle size of the inner phase. The mean particle size (Sauterdiameter, D[3, 2]) of the inner phase of the emulsion was 440 nm asmeasured by laser diffraction (Malvern Masersizer).

d) Preparation of a Solid Formulation from the Emulsion:

The emulsion may be sprayed into a pre-cooled fluidised bed ofcornstarch. Excess cornstarch can be removed by sieving and the powderobtained can be dried in an air stream at room temperature. The powderparticle fraction in the range of 0.16 to 0.63 mm can be collected bysieving and characterised with respect to the carotenoid content, thecolour intensity and the colour hue in an aqueous dispersion, thecontent of the corn starch and residual humidity.

TABLE 8 Calculated composition of the dried formulation Amount[weight-%, based on the total Compound dry weight] RP_(Glu): Riceendosperm 25.0 protein according to example 9 sucrose 30.5 ascorbylpalmitate 1.5 β-carotene 11.5 corn oil 5.5 dl-α-tocopherol 1.0 Cornstarch fluid 25.0

Example 19 Manufacture of a Formulation of β-Carotene

A formulation comprising a rice endosperm protein and (3-carotene may beprepared as follows:

a) Preparation of a(n Oil-Based) Solution 1:

6.6 g of corn oil and 1.2 g of dl-α-tocopherol were mixed. 13.8 g ofcrystalline (3-carotene were dispersed in 180 ml of chloroform(trichloromethane) and the resulting dispersion was added to the mixtureof corn oil and tocopherol. By gently stirring and simultaneous heatingthe mixture to about 60° C. a solution was obtained.

b) Preparation of a(n Aqueous) Solution 2:

30 g of RP_(XG) (Glycosylation of rice endosperm protein with Xanthangum) according to Example 10 was re-dissolved in 120 ml of water bystirring at 60° C. Additionally 1.8 g of ascorbyl palmitate and 36.6 gof sucrose were added. 0.5 ml of aqueous 1 N NaOH were used to adjustthe pH to a value of 7.3.

e) Preparation of an Emulsion from the Solutions 1 and 2:

Under vigorous stirring solution 1 was added to solution 2 at 53° C. andthe dispersion was vigorously stirred for another 30 minutes. Thestirred dispersion was kept at 50 to 55° C. for 30 minutes. Residualtrichloromethane was removed at 50 to 55° C. After removing entrappedair bubbles by centrifugation the emulsion was gently stirred at 50 to55° C. for some minutes and then characterised with respect to theparticle size of the inner phase. The mean particle size (Sauterdiameter, D[3, 2]) of the inner phase of the emulsion was 380 nm asmeasured by laser diffraction (Malvern Masersizer).

d) Preparation of a Solid Formulation from the Emulsion:

The emulsion may be sprayed into a pre-cooled fluidised bed ofcornstarch. Excess cornstarch can be removed by sieving and the powderobtained can be dried in an air stream at room temperature. The powderparticle fraction in the range of 0.16 to 0.63 mm can be collected bysieving and characterised with respect to the carotenoid content, thecolour intensity and the colour hue in an aqueous dispersion, thecontent of the corn starch and residual humidity.

TABLE 9 Calculated composition of the dried formulation Amount[weight-%, based on the total Compound dry weight] RP_(XG): Riceendosperm 25.0 protein according to example 10 sucrose 30.5 ascorbylpalmitate 1.5 β-carotene 11.5 corn oil 5.5 dl-α-tocopherol 1.0 Cornstarch fluid 25.0

Example 20 Manufacture of a Formulation of β-Carotene

A formulation comprising a rice endosperm protein and β-carotene may beprepared as follows:

a) Preparation of a(n Oil-Based) Solution 1:

6.6 g of corn oil and 1.2 g of dl-α-tocopherol were mixed. 13.8 g ofcrystalline β-carotene were dispersed in 180 ml of chloroform(trichloromethane) and the resulting dispersion was added to the mixtureof corn oil and tocopherol. By gently stirring and simultaneous heatingthe mixture to about 60° C. a solution was obtained.

b) Preparation of a(n Aqueous) Solution 2:

30 g of RP_(DA) (Deamidation of rice endosperm protein using alkalitreatment) according to Example 16 was re-dissolved in 120 ml of waterby stirring at 60° C. Additionally 1.8 g of ascorbyl palmitate and 36.6g of sucrose were added. 0.4 ml of aqueous 1 N NaOH were used to adjustthe pH to a value of 7.0.

c) Preparation of an Emulsion from the Solutions 1 and 2:

Under vigorous stirring solution 1 was added to solution 2 at 53° C. andthe dispersion was vigorously stirred for another 30 minutes. Thestirred dispersion was kept at 50 to 55° C. for 30 minutes. Residualtrichloromethane was removed at 50 to 55° C. After removing entrappedair bubbles by centrifugation the emulsion was gently stirred at 50 to55° C. for some minutes and then characterised with respect to theparticle size of the inner phase. The mean particle size (Sauterdiameter, D[3, 2]) of the inner phase of the emulsion was 500 nm asmeasured by laser diffraction (Malvern Masersizer).

d) Preparation of a Solid Formulation from the Emulsion:

The emulsion may be sprayed into a pre-cooled fluidised bed ofcornstarch. Excess cornstarch can be removed by sieving and the powderobtained can be dried in an air stream at room temperature. The powderparticle fraction in the range of 0.16 to 0.63 mm can be collected bysieving and characterised with respect to the carotenoid content, thecolour intensity and the colour hue in an aqueous dispersion, thecontent of the corn starch and residual humidity.

TABLE 10 Calculated composition of the dried formulation Amount[weight-%, based on the total Compound dry weight] RP_(DA): Riceendosperm 25.0 protein according to example 14 sucrose 30.5 ascorbylpalmitate 1.5 β-carotene 11.5 corn oil 5.5 dl-α-tocopherol 1.0 Cornstarch fluid 25.0

1-36. (canceled)
 37. A process for the manufacture of a rice endospermprotein which is partially deamidated starting from milled rice, wherebybefore milling the rice bran was removed, comprising the following stepsa) to e): a) preparing an aqueous solution or suspension of milled rice,whereby the rice bran was removed before milling, whereby the solutionor suspension preferably has a dry mass content of from 0.1 to 30weight-%, based on the total amount of the aqueous solution orsuspension; b) optionally removing the non-protein part or the proteinpart of the milled rice, whereby the rice bran was removed beforemilling, to obtain the rice endosperm protein; c) modifying the proteinpart of the milled rice, whereby the rice bran was removed beforemilling, by partially deamidating the protein part of the milled rice toobtain rice endosperm protein which is partially deamidated; d)optionally isolating the rice endosperm protein which is partiallydeamidated; and e) optionally converting the rice endosperm proteinwhich is partially deamidated into a solid form, wherein the riceendosperm protein has an emulsion activity of ≧0.62 as determinedturbidimetrically by emulsifying a solution of a sample rice endospermprotein in corn oil, homogenized by sonication and measuring absorbance,the absorbance at a time 0 after homogenization indicating the emulsionactivity of the rice endosperm.
 38. The process according to claim 37,wherein the removal of the non-protein part (step b) is achieved bytreating the milled rice with non-protein degrading enzymes,deactivating the enzymes, separating and removing the non-protein partfrom the protein part of the milled rice.
 39. The process according toclaim 38, wherein the non-protein degrading enzymes are starch-degradingenzymes, cellulose-degrading enzymes or mixtures thereof.
 40. Theprocess according to claim 38, wherein the separation of the non-proteinpart is achieved by centrifugation followed by washing off thenon-protein part with water.
 41. The process according to claim 37,wherein the removal of the protein part (step b) is achieved byadjusting the pH of the milled rice solution or suspension to a value offrom 7 to
 12. 42. The process according to claim 37, wherein step e) isachieved by drying.
 43. The process according to claim 37, wherein thedeamidation is achieved by adjusting the pH value of an aqueouscolloidal solution of the protein part of the rice endosperm proteinobtained in step b) to a value in the range of from 9.0 to 13.0 and at atemperature in the range of from 25 to 90° C.
 44. A rice endospermprotein which is partially deamidated obtainable by any processaccording to claim
 37. 45. A composition comprising a rice endospermprotein obtained by the process of claim 37 and a fat-soluble activeingredient and/or a fat-soluble colorant.
 46. The composition accordingto claim 45, wherein the fat-soluble active ingredient and/or thefat-soluble colorant is a carotene or a structurally related polyenecompound, a fat soluble vitamin, a triglyceride rich in polyunsaturatedfatty acids, an oil soluble UV-A filter, an UV-B filter or a mixturethereof.
 47. The composition according to claim 46, wherein the caroteneor structurally related polyene compound is a carotenoid such asα-carotene, β-carotene, 8′-apo-β-carotenal, 8′-apo-β-carotenoic acidesters, canthaxanthin, astaxanthin, lycopene, lutein, zeaxanthin,crocetin, α-zeacarotene, β-zeacarotene or a mixture thereof.
 48. Thecomposition according to claim 47, wherein the carotenoid is β-carotene.49. The composition as in claim 46, wherein the fat-soluble vitamin isVitamin A or E.
 50. The composition as in claim 45, wherein at least onecompound selected from the group consisting of monosaccharides,disaccharides, oligosaccharides, polysaccharides, glycerol,triglycerides, water-soluble antioxidants and fat-soluble antioxidantsis additionally present.
 51. The composition as in claim 50, wherein themono- or disaccharide is sucrose, invert sugar, xylose, glucose,fructose, lactose, maltose, saccharose and sugar alcohols.
 52. Thecomposition as in claim 50, wherein the oligo- or polysaccharide is astarch, a starch hydrolysate or a modified starch.
 53. The compositionas in claim 52, wherein the starch hydrolysate is a dextrin, amaltodextrin or a glucose syrup.
 54. The composition as in claim 46,wherein the triglyceride is a vegetable oil or fat.
 55. The compositionas in claim 45, wherein a co-emulgator selected from the groupconsisting of mono- and diglycerides of fatty acids, polyglycerol estersof fatty acids, lecithins, and sorbitan monostearate is additionallypresent.
 56. The composition as in claim 45, wherein the rice endospermprotein has an emulsion activity of ≧0.6.
 57. The composition as inclaim 45, wherein the rice endosperm protein has an emulsion stabilityof ≧20 minutes.
 58. The composition as in claim 45, wherein the amountof the rice endosperm protein which is partially deamidated is fromabout 1 to about 70 weight-% and/or the amount of the fat-soluble activeingredient and/or the fat-soluble colorant is from about 0.1 to about 90weight-%, based on the total amount of the composition.
 59. Thecomposition as in claim 45 in the form of a powder.
 60. A process forthe manufacture of a composition as claimed in claim 45 which comprisesthe following steps: I) preparing an aqueous solution or colloidalsolution of a rice endosperm protein which is partially deamidated, II)optionally adding at least a water-soluble excipient and/or adjuvant tothe solution prepared in step I), III) preparing a solution ordispersion of at least a fat-soluble active ingredient and/or afat-soluble colorant, and optionally at least a fat-soluble adjuvantand/or excipient, IV) mixing the solutions prepared in step I) to III)with each other, V) homogenising the thus resulting mixture, VI)optionally adding a cross-linking agent for (further) cross-linking therice endosperm protein which is partially deamidated, VIa) optionallysubmitting the mixture resulting after having performed step VI) toenzymatic treatment or heat treatment to cross-link the modified riceendosperm protein VII) optionally converting the dispersion obtained instep V) and/or VI) into a powder, VIII) optionally drying the powderobtained in step VII), IX) optionally submitting the powder resultingfrom step VII or the dry powder resulting from step VIII to heattreatment or to enzymatic treatment to cross-link the rice endospermprotein which is partially deamidated, with the proviso that only stepVIa) or step IX) is carried out, but not both, when step VI) is carriedout.
 61. The process according to claim 60, wherein the enzymatictreatment according to step VIa) or step IX) is a treatment with across-linking enzyme.
 62. The process according to claim 60 wherein therice endosperm protein which is partially deamidated is one having anemulsion activity of ≧0.6.
 63. Food, beverages, animal feed, personalcare and pharmaceutical compositions containing a composition as claimedin claim
 45. 64. Food, beverages, animal feed, personal care andpharmaceutical compositions containing a rice endosperm protein asclaimed in claim
 44. 65. Process for the protection of fat-solubleactive ingredients and/or fat-soluble colorants by whereby the riceendosperm protein as claimed in claim 44 is added to said fat-solubleactive ingredients and/or said fat-soluble colorants as a protectingagent.
 66. The process according to claim 42, wherein the drying is byfreeze drying or spray drying.
 67. The composition as in claim 42,wherein the rice endosperm protein has an emulsion activity of ≧0.62.68. The composition as in claim 45, wherein the rice endosperm proteinhas an emulsion activity of ≧0.7.
 69. The composition as in claim 45,wherein the rice endosperm protein has an emulsion activity of ≧23minutes.
 70. The composition as in claim 45, wherein the rice endospermprotein has an emulsion activity of ≧25 minutes.
 71. The processaccording to claim 61, wherein the cross-linking enzyme is atransglutaminase.
 72. Process for the enrichment, fortification and/orcoloration of food, beverages, animal feed, personal care orpharmaceutical compositions, whereby a composition as claimed in claim45 is added to the food, beverages, animal feed, personal care andpharmaceutical compositions, respectively.
 73. A process for themanufacture of a rice endosperm protein which is partially deamidatedstarting from milled rice, whereby before milling the rice bran wasremoved, comprising the following steps a) to e): a) preparing anaqueous solution or suspension of milled rice, whereby the rice bran wasremoved before milling, whereby the solution or suspension preferablyhas a dry mass content of from 0.1 to 30 weight-%, based on the totalamount of the aqueous solution or suspension; b) optionally removing thenon-protein part or the protein part of the milled rice, whereby therice bran was removed before milling, to obtain the rice endospermprotein; c) modifying the protein part of the milled rice, whereby therice bran was removed before milling, by partially deamidating theprotein part of the milled rice to obtain rice endosperm protein whichis partially deamidated; d) optionally isolating the rice endospermprotein which is partially deamidated; and e) optionally converting therice endosperm protein which is partially deamidated into a solid formwherein the rice endosperm protein has an emulsion stability of ≧23minutes where emulsion stability is calculated by the formula: To×Δ/Δ,where ΔT is the decrease in turbidity (absorbance) of an initialabsorbance (To) over/after a time interval of Δt 10 minutes.